Project Summary The project aims to develop an HIV nonhuman primate model in which the breadth of protection by HIV Env- based active or passive immunizations can be determined in a quantitative manner in a limited number of animals. This should greatly improve the applicability of NHP studies for optimization and selection of candidate HIV vaccines or monoclonal antibodies for clinical efficacy studies and thus help to accelerate HIV vaccine development. The number of animals needed to determine the breadth of protection will be minimized by i) reducing animal to animal variation through referencing to an internal challenge virus control, ii) limiting the efficacy measurement to a single round of replication, and iii) challenging each animal simultaneously with a panel of different challenge viruses. The SIV based challenge viruses use HIV-1 Env for entry into target cells during the first replication cycle, but then switch to SIV Env for all subsequent rounds of infection. A challenge virus that uses SIV Env during the first and all subsequent rounds is included as an internal control that is not affected by HIV Env antibodies. Genetic tagging and next generation sequencing allows to determine the ratios of the co-injected challenge viruses and the influence of HIV Env antibodies on these ratios. As a proof of concept, fifteen different genetic tags will be introduced into the challenge virus and explored for their affect on virus replication. Based on these tags, a panel of challenge viruses will be generated and characterized that use 12 HIV Envs derived from transmitted founder viruses of different clades and geographic areas together with the internal control virus. Determination of the ratio of the HIV Env challenge virus to the internal control virus will be determined in rhesus PBMC cultures. To demonstrate in vivo infectivity of the 12 different HIV Env challenge viruses, they will be co-inoculated with the internal control virus intrarectally into six rhesus macaques. Ten days after inoculation the ratio of each HIV Env challenge virus to the internal control virus will be determined in the blood and different lymphatic tissues. The variation in the ratios between different animals should allow to determine group sizes needed in subsequent challenge experiments after active or passive immunization.